Our group has continued studies of chromatin structure and the regulation of eukaryotic gene expression by analysis of several ATP-dependent chromatin remodeling enzymes. This year we have further elucidated the biology of ATP-dependent chromatin remodeling by NURF (Nucleosome Remodeling Factor) in the mouse. We have extended analysis of mutants for Bptf, the largest subunit of NURF, by studying ES cells deficient for that subunit. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcription factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. A manuscript describing these findings is being revised for publication. We are also making progress on studies of the budding yeast multi-protein SWR1 complex, a member of the SWI2/SNF2 superfamily of chromatin remodeling enzymes. Previously, we established a new link between the SWR1 complex and the histone variant H2AZ, which is incorporated preferentially at gene promoter regions. We showed that deposition of histone H2AZ is mediated by the SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. We are in the process of analyzing the early steps of histone replacement by a number of biochemical assays, which should provide insight into histone exchange mechanism. We have also continued studies of the Scm3 nonhistone protein, which we previously found to interact with the centromeric histone variant CenH3, and to be required for assembly of CenH3 and the inner kinetochore in vivo. We are currently undertaking in vivo studies of Scm3 and CenH3 deposition in the cell cycle, and in vitro reconstitution of Scm3-CenH3-H4 complexes with centromeric DNA. We have continued fruitful collaborations with intramural and extramural colleagues. Several manuscripts reporting these collaborative studies were published in the past year.